Amino-acid sequence of toxin III of Naja haje.

The complete amino acid sequence of toxin III of Naja haje (72 residues) has been established mainly by use of a protein sequenator (identification of 70 residues). The two C-terminal residues have been determined by digestion with carboxypeptidases A and B. Addition of succinylated protein or peptide greatly improved the performance of the sequenator for the Edman degradation of peptides: on one peptide (39 residues) degradation went to step 34 with a protein program and on two peptides (10 and 13 residues) degradation reached the last amino acid with a peptide program (use of dimethylbenzylamine). Amino acid analysis of tryptic peptides obtained by digestion of the C-terminal cyanogen bromide peptide are in full agreement with the sequence established by automatic degradation. The sequence of toxin III of Naja haje is unique and is very similar to that of Naja nivea alpha (although there are 9 differences), of Naja melanoleuca b (11 differences) and also to that of Naja naja A (18 differences).     

Experimental study on developing rat kidney after Naja haje envenomation

In the present experiment 164 pregnant white Wistar rats were used to study the effect of Naja haje (Egyptian cobra) venom on the developing kidney. The rats were divided into 3 groups; a control group, a group receiving one LD50 of N. haje venom and the third injected with 1/8 of LD50. The injection was performed at different stages of gestation. After birth, the neonates of group I and III and embryos of group II were examined histologically, histochemically and electron-microscopically. Both lethal and sublethal doses of N. haje venom produced haemorrhages and vascular congestion of the developing kidney. The lethal dose had degenerative effects on the podocytes and endothelium. Tubular damage appeared mainly as mitochondrial degeneration and bud-like extension, protrusions of cytoplasm and vacuolization. The succinic dehydrogenase enzyme showed decreased activity. The sublethal dose had an effect on the glomerular basement membrane in the form of splitting, increased mesangial cells and matrix, mitochondrial degeneration and fusion of podocyte processes. Tubulization of the parietal epithelium, vacuolization of the proximal tubules, mitochondrial degeneration and apical budding were evident.     

The amino acid sequence of toxin V II 2, a cytotoxin homologue from banded Egyptian cobra (Naja haje annulifera) venom.

Toxin V II 2 comprises 60 amino acid residues and is cross-linked by four disulphide bridges. The complete amino acid sequence of this toxin was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides were purified by ion-exchange chromatography and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequenator or by manual manipulation, was employed to obtain the sequence of the intact toxin and the pure peptides. The chymotryptic digest provided the necessary overlapping peptides which allowed the alignment of tryptic peptides. The amino acid sequence of Naja haje annulifera toxin V II 2 shows a high degree of homology with cytotoxin V II 1 of the same venom.     

Comparative Study of Structure and Activity of Cytotoxins from Venom of the Cobras Naja oxiana, Naja kaouthia, and Naja haje

Cytotoxins are positively charged polypeptides that constitute about 60% of all proteins in cobra venom; they have a wide spectrum of biological activities. By CD spectroscopy, cytotoxins CT1 and CT2 Naja oxiana, CT3 Naja kaouthia, and CT1 and CT2 Naja haje were shown to have similar secondary structure in an aqueous environment, with dominating beta-sheet structure, and to vary in the twisting angle of the beta-sheet and the conformation of disulfide groups. Using dodecylphosphocholine micelles and liposomes, CT1 and CT2 Naja oxiana were shown to incorporate into lipid structures without changes in the secondary structure of the peptides. The binding of CT1 and CT2 Naja oxiana with liposomes was associated with an increase in the beta-sheet twisting and a sign change of the dihedral angle of one disulfide group. The cytotoxins were considerably different in cytotoxicity and cooperativity of the effect on human promyelocytic leukemia cells HL60, mouse myelomonocytic cells WEHI-3, and human erythroleukemic cells K562. The most toxic CT2 Naja oxiana and CT3 Naja kaouthia possessed low cooperativity of interaction (Hill coefficient h = 0.6-0.8), unlike 10-20-fold less toxic CT1 and CT2 Naja haje (h = 1.2-1.7). CT1 Naja oxiana has an intermediate position on the cytotoxicity scale and is characterized by h = 0.5-0.8. The cytotoxins under study induced necrosis of HL60 cells and failed to activate apoptosis. The differences in cytotoxicity are supposed to be related not with features of the secondary structure of the peptides, but with interactions of side chains of variable amino acid residues with lipids and/or membrane proteins.     

The embryonic development of the Egyptian cobra Naja h. haje (Squamata: Serpentes: Elapidae)

The Egyptian cobra, Naja h. haje, is the largest of the African cobras and is a member of a successful and medically important species complex found throughout Africa, north and south of the Sahara, as well as across the Arabian Peninsula to Oman. Although its phylogenetic position and venom characteristics have been well studied, its development has not. Here, we present a normal staging table for N. h. haje, based on external features. Comparison with firstly the Asian monocled cobra, Naja kaouthia, and then with the small number of other oviparous snake species, allowed us to examine whether differences between two species in the same genus were of the same type and magnitude as those between unrelated genera. In fact, at least with respect to external features, we found a similar level of disparity. N. h. haje embryos lagged behind those of N. kaouthia in body and head scale development, size in ovo and hatchling length, despite having a slightly shorter incubation period and a somewhat larger adult size. Some of these differences may have been the result of differing incubation temperatures. Nonetheless, there does appear to be a broadly conserved pattern of in ovo development in at least macrostomatan snakes.     

The purification and amino acid sequence of toxin CM-13b from Naja haje annulifera (Egyptian cobra) venom.

Toxin CM-13b was purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. The toxin comprises 65 amino acid residues and is cross-linked by five disulphide bridges. The complete amino acid sequence of toxin CM-13b was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides purified by DEAE-cellulose chromatography and chromatography or electrophoresis on paper. The amino acid sequences of the intact toxin and its constituent peptides were determined by the Edman-Begg procedure, either through the use of the automatic sequenator or by manual manipulation. The chymotryptic digest provided the necessary overlapping peptides for aligning the tryptic peptides. The primary structure of toxin CM-13b shows a high degree of homology with that of protein S4C11 from Naja melanoleuca venom[1], but their toxicities are very different.     

Snake venom toxins. The amino acid sequence of three toxins (CM-2e, CM-4a and CM-) from Naja haje annulifera (Egyptian cobra) venom.

Three toxins (CM-2e, CM-4a and CM-7) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex and by ion-exchange chromatography on CM-cellulose. They comprise 60 amino acid residues and are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of the three toxins have been elucidated. The toxicities, the serological properties, the sequences and the invariant amino acid residues of toxin CM-2e, CM-4a and CM-7 resemble the corresponding properties of the cytotoxin group.     

Snake venom toxins. The amino-acid sequences of three toxins (CM-8, CM-11 and CM-13a) from Naja haje annulifera (Egyptian cobra) venom.

Three toxins (CM-8, CM-11, and CM-13a) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. Whereas toxin CM-8 and CM-11 comprise 60 amino acid residues, toxin CM-13a contains 61 residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have been elucidated. The reduced and S-carboxymethylated toxins were digested with trypsin and chymotrypsin and the peptides purified by ion-exchange chromatography, gel filtration and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequencer or by manual manipulation, was employed to obtain the sequence of the intact toxins and the pure peptides. The chymotryptic digests provided the necessary overlapping peptides which allowed the alignment of the tryptic peptides. The properties of the three toxins were compared with those of the cytotoxin group. The toxicities the serological properties, the sequences and the invariant amino acid residues of toxin CM-8 and CM-11 resemble the corresponding properties of the cytotoxin group. The sequence and serological properties of toxin CM-13a show that it is related to the cytotoxin group, but its toxicity is much lower than those encountered in the cytotoxin group.     

Serum catecholamine and hormonal titers in the hibernating snake Naja haje haje, with reference to the annual climatic cycle

(1) The analysis of catecholamines revealed alterations in norepinephrine levels in the blood serum and the central nervous system of the Egyptian cobra during the different phases of the hibernation cycle. This may reflect an increased central demand for norepinephrine as a mediator for adaptive changes to cold.

(2) The low and variable concentration of dopamine suggests retarded synthesis during all the phases studied.

(3) Serotonin may be depleted or associated with increased activity of serotonergic neurons during cold acclimation. This increases the rate of turnover of serotonin to 5-hydroxy indole acetic acid.

(4) Cricannual measurements of cortisol and adrenocorticotrophic hormones revealed marked suppression that would clarify the direct effect of hibernation on higher centers. The decreased levels during prehibernation and hibernation were restored in normothermic animals when the pituitary–adrenal axis resumed its activity.

(5) The decline in thyroid hormone concentrations has its behavioural and physiological responses that lead to seasonal adjustments in the hibernating cobras.

Snake alpha-neurotoxin binding site on the Egyptian cobra (Naja haje) nicotinic acetylcholine receptor Is conserved

Evolutionary success requires that animal venoms are targeted against phylogenetically conserved molecular structures of fundamental physiological processes. Species producing venoms must be resistant to their action. Venoms of Elapidae snakes (e.g., cobras, kraits) contain alpha-neurotoxins, represented by alpha-bungarotoxin (alpha-BTX) targeted against the nicotinic acetylcholine receptor (nAChR) of the neuromuscular junction. The model which presumes that cobras (Naja spp., Elapidae) have lost their binding site for conspecific alpha-neurotoxins because of the unique amino acid substitutions in their nAChR polypeptide backbone per se is incompatible with the evolutionary theory that (1) the molecular motifs forming the alpha-neurotoxin target site on the nAChR are fundamental for receptor structure and/or function, and (2) the alpha-neurotoxin target site is conserved among Chordata lineages. To test the hypothesis that the alpha-neurotoxin binding site is conserved in Elapidae snakes and to identify the mechanism of resistance against conspecific alpha-neurotoxins, we cloned the ligand binding domain of the Egyptian cobra (Naja haje) nAChR alpha subunit. When expressed as part of a functional Naja/mouse chimeric nAChR in Xenopus oocytes, this domain confers resistance against alpha-BTX but does not alter responses induced by the natural ligand acetylcholine. Further mutational analysis of the Naja/mouse nAChR demonstrated that an N-glycosylation signal in the ligand binding domain that is unique to N. haje is responsible for alpha-BTX resistance. However, when the N-glycosylation signal is eliminated, the nAChR containing the N. haje sequence is inhibited by alpha-BTX with a potency that is comparable to that in mammals. We conclude that the binding site for conspecific alpha-neurotoxin in Elapidae snakes is conserved in the nAChR ligand binding domain polypeptide backbone per se. This conclusion supports the hypothesis that animal toxins are targeted against evolutionarily conserved molecular motifs. Such conservation also calls for a revision of the present model of the alpha-BTX binding site. The approach described here can be used to identify the mechanism of resistance against conspecific venoms in other species and to characterize toxin-receptor coevolution.     

Moringa oleifera leaf fractions attenuated Naje haje venom-induced cellular dysfunctions via modulation of Nrf2 and inflammatory signalling pathways in rats

Naja haje envenoming could activate multiple pathways linked to haematotoxic, neurological, and antioxidant systems dysfunctions. Moringa oleifera has been used in the management of different snake venom-induced toxicities, but there is no scientific information on its antivenom effects against Naja haje. This study thus, investigated the antivenom activities of different extract partitions of M. oleifera leaves against N. haje envenoming. Forty five male rats were divided into nine groups (n = 5). Groups 2 to 9 were envenomed with 0.025 mg/kg (LD₅₀) of N. haje venom while group 1 was given saline. Group 2 was left untreated, while group 3 was treated with polyvalent antivenom, groups 4, 6 and 8 were treated with 300 mg/kg^?1 of N-hexane, ethylacetate and ethanol partitions of M. oleifera, respectively. Groups 5, 7 and 9 were also treated with 600 mgkg^?1of the partitions, respectively. Ethanol extract and ethyl acetate partition of M. oleifera significantly improved haematological indices following acute anaemia induced by the venom. Likewise, haemorrhagic, haemolytic and anti-coagulant activities of N. haje venom were best inhibited by ethanol partition. Envenoming significantly down-regulated Nuclear factor erythroid 2-related factor 2 (Nrf2) with the consequent elevation of antioxidant enzymes activities in the serum and brain. Treatment with extract partitions however, elevated Nrf2 levels while normalising antioxidant enzyme activities. Furthermore, there were reduction in levels of pro-inflammatory cytokines (TNF-α and interleukin-1β) in tissues of treated envenomed rats. This study concludes that ethanol partition of M. oleifera was most effective against N. haje venom and could be considered as a potential source for antivenom metabolites.